This invention relates to the design and synthesis of certain 2- and 4-substituted pyrimidine nucleosides that are useful for incorporation into oligonucleotides. The oligonucleotides, in turn, are useful as diagnostic reagents, research reagents and in therapeutics.
It is well known that most of the bodily states in multicellular organisms, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect might be obtained with minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression which would lead to undesired protein formation.
One method for inhibiting specific gene expression is with the use of oligonucleotides. Oligonucleotides complementary to a specific target messenger RNA (mRNA) sequence are used. Several oligonucleotides are currently undergoing clinical trials for such use.
Transcription factors interact with double-stranded DNA during regulation of transcription. Oligonucleotides can serve as competitive inhibitors of transcription factors to modulate the action of transcription factors. Several recent reports describe such interactions (see, Bielinska, et. al., Science 1990, 250, 997-1000; and Wu, et al., Gene 1990, 89, 203-209.)
Oligonucleotides also have found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with the above gene expression inhibition, diagnostic use can take advantage of an oligonucleotides ability to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligonucleotides via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are said to be complementary to one another.
Oligonucleotides are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of such other biological molecules. One particular use, the use of oligonucleotides as primers in the reactions associated with polymerase chain reaction (PCR), has been the cornerstone for the establishment of an ever expanding commercial business. The use of such PCR reactions has seemingly xe2x80x9cexplodedxe2x80x9d as more and more use of this very important biological tool are practiced. The uses of PCR have extended into many areas in addition to those contemplated by its nobel laureate inventor. Examples of such new areas include forensics, paleontology, evolutionary studies and genetic counseling to name just a few. Primers are needed for each of these uses. Oligonucleotides, both natural and synthetic, serve as the primers.
Oligonucleotides also are used in other laboratory procedures. A number of these uses are described in common laboratory manuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; and Current Protocols In Molecular Biology, F. M. Ausubel, et. al., Eds., Current Publications, 1993. Such uses include Synthetic Oligonucleotide Probes, Screening Expression Libraries with Antibodies and Oligonucleotides, DNA Sequencing, In Vitro Amplification of DNA by the Polymerase Chain Reaction and Site-directed Mutagenesis of Cloned DNA from Book 2 of Molecular Cloning, A Laboratory Manual, ibid. and DNA-Protein Interactions and The Polymerase Chain Reaction from Vol. 2 of Current Protocols In Molecular Biology, ibid.
To supply the users of oligonucleotides, many scientific journals now contain advertisements for either oligonucleotide precursors or for custom-synthesized oligonucleotides. This has become an important commercial use of oligonucleotides. Other oligonucleotides can be synthesized to have properties that are tailored for the desired use. Thus, a number of chemical modifications have been introduced into oligonucleotides to increase their usefulness in diagnostics, as research reagents, and as therapeutic entities. These modifications are designed to increase binding to a target strand, to assist in identification of the oligonucleotide or an oligonucleotide-target complex, to increase cell penetration, to provide stability against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonuclectides, to provide a mode of disruption (terminating event) once sequence-specifically bound to a target, or to improve the pharmacokinetic properties of the oligonucleotides.
To serve some of the above-noted uses, reactive functionalities can be tethered to an oligonucleotide and directed at phosphodiester and heterocyclic centers in target compounds. The positioning of these functionalities may require the synthesis of C-2 and C-4 substituted pyrimidine nucleosides with the following general features: (a) preservation of Watson-Crick hybridization characteristics; (b) stability during automated DNA synthesis; and (c) the conformational rigidity of a carbon tether to accurately place functionalities near their target. Criteria (a) and (b) are not met by readily available 2-O-, 2-S-, or 2-N-alkyl pyrimidine nucleosides since these groups are sensitive to nucleophilic displacement. For example, deprotecting DNA oligomers containing S-alkylated moieties with concentrated ammonia likely would yield displacement products at C-2 and C-4 pyrimidine positions. In addition, 2-O-, 2-S-, and 2-N-alkyl groups would preclude the availability of an imino proton at N-3, which is necessary for hydrogen bonding. Likewise, nucleosides having 2-N-alkyl groups at these positions would fail to meet criteria (b) and (c) due to their chemical instability and the spatial instability inherent in an inversion of configuration of a tertiary nitrogen. Accordingly, there remains a need in the art for novel 2- and 4-substituted pyrimidines that meet the design criteria set forth above.
It is one object of this invention to provide oligonucleotides for use in diagnostics, therapeutics and as research reagents.
It is another object of this invention to provide oligonucleotides that effectively modulate the activity of RNA or DNA or proteins.
It is a further object to provide novel 2-substituted pyrimidines.
These and other objects will become apparent to persons of ordinary skill in the art from a review of the present specification and appended claims.
In accordance with these and other objects, the present invention provides novel C-2 and C-4 modified pyrimidine nucleosides and oligonucleosides. In certain embodiments, the compounds of the invention have structure 1 or structure 2: 
wherein:
J is N or CH;
R5 is H or CH3;
one of R2 and R4 is xe2x95x90O, xe2x95x90NH, or xe2x95x90NH2+; and the other of R2 and R4 is Q, xe2x95x90C(RA)xe2x80x94Q, C(RA) (RB)xe2x80x94C(RC) (RD)xe2x80x94Q, C(RA)xe2x95x90C(RC)xe2x80x94Q or Cxe2x89xa1Cxe2x80x94Q;
RA, RB, RC and RD independently are H, SH, OH, NH2, or C1-C20 alkyl, or one of (RA) (RB) or (RC) (RD) is xe2x95x90O;
Q is halogen, hydrogen, C1-C20 alkyl, C1-C20 alkylamine, C1-C20 alkyl-N-phthalimide, C1-C20 alkylimidazole, C1-C20 alkylbis-imidazole, imidazole, bis-imidazole, amine, N-phthalimide, C2-C20 alkenyl, C2-C20 alkynyl, hydroxyl, thiol, keto, carboxyl, nitrates, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, O-(hydroxyl protecting group), a leaving group, a heterocycle, an intercalator, a reporter molecule, a conjugate, a polyamine, a polyamide, a polyethylene glycol, a polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, a group that enhances the pharmacokinetic properties of oligonucleotides, a RNA cleaving moiety or a depurination enhancing group;
T3 and T5 independently are H, phosphate, an activated phosphate, a hydroxyl protecting group, a nucleoside, a nucleotide, an oligonucleotide or an oligonucleoside;
X1 and X2 independently are hydrogen, halogen, hydroxyl, O-(hydroxyl protecting group), thiol, carboxyl, nitrate, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, heterocyclic, alicyclic, carbocyclic, intercalators, reporter molecules, conjugates, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligonucleotides, or groups that enhance the pharmacokinetic properties of oligonucleotides.
For structure 1, when T5 is a hydroxyl protecting group or together T3 and one of X1 or X2 is a hydroxyl protecting group, then R2 should not be CH3; when T3 and T5 are OH or O-benzoyl, then R2 should not be CH2OC6H5; and when R4 is Q then Q is not hydrogen, hydroxyl, thiol, amino or hydrogen.
The present invention also provides methods for preparing the compounds of the invention and synthetic intermediates employed in the methods. In certain embodiments, the methods involve reacting 2,2xe2x80x2- or 2,5xe2x80x2-anhydropyrimidines with a nucleophile to effect attack of the nucleophile at the 2-pyrimidine position. The nucleophile can have general formula R2 or can be a functional group that can be modified to have formula R2. In further embodiments, the methods involve reacting 4-halopyrimidines with substituted alkynes in the presence of an organometallic coupling reagent for a time and under reaction conditions effective to form a covalent bond between the alkyne and the pyrimidine 4-position.
The invention also provides methods for modulating the production or activity of a protein in an organism comprising contacting the organism with a compound according to the invention. It is preferred that the compound include a base sequence that is complementary to a RNA or DNA sequence that codes for the protein. This xe2x80x9ctargeting portionxe2x80x9d of the compound is, thus selected to be complementary to the targeted portion of DNA or RNA such that the compound can bind to the target portion.